Chromatin Structure and Gene Expression

Year: 1976 DOI: 10.7907/3ayy-cg55
Rat-liver chromatin has been separated into nuclease-sensitive and resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg⁺⁺-soluble and insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min under standard conditions), 11% of the input chromatin DNA is found in the Mg⁺⁺-soluble fraction. This DNA has a weight-average single strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of middle repetitive and nonrepetitive DNA sequences of the rat genome. Cross-reassociation experiments show that a fractionation of whole genomal DNA sequences has been achieved. Moreover, the Mg⁺⁺-soluble fraction of liver chromatin is enriched in nonrepeated sequences coding for liver RNA but not for brain RNA. Fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained. The Mg⁺⁺-soluble, template-active fraction is enriched five-fold in DNA sequences complementary to RNA.

The Mg⁺⁺-soluble fraction is enriched in nonhistone chromosomal proteins and depleted in histone protein. Histone I (fl) is absent from the Mg⁺⁺-soluble active fraction. About half of the DNA of both Mg⁺⁺-soluble and Mg⁺⁺-insoluble fractions is resistant to prolonged digestion with DNAase II or staphylococcal nuclease. The nuclease- resistant structures of inactive (Mg⁺⁺-insoluble) chromatin are DNAhistone complexes which sediment at 11-13S. Two nuclease-resistant species are present in active chromatin. These particles sediment at 15 and 20S, respectively, and contain DNA, RNA, histone and nonhistone proteins. Thermal denaturation studies suggest that the· DNA of active chromatin is complexed primarily with nonhistone proteins.